At any time, cells need to be able to respond to environmental stressors that are capable of inducing DNA damage. Various protective mechanisms are in place to ensure that DNA damage is repaired and not passed on to the next generation of cells. One of the key molecules involved in this process is the protein p53. When p53 status is compromised, the cell is left unable to cope with environmental stress and the replication of cells with damaged DNA results in cancer, the second greatest cause of mortality in the United States. Previous results in our lab suggest that p53 signalling, in response to DNA damage, may involve sphingolipids. Sphingolipids are important regulators of cell growth and death. While the bioactive lipids ceramide and sphingosine act as pro-death molecules, sphingoslne-1-phosphate (SIP) has been shown to stimulate proliferation and angiogenesis. The primary enzyme regulating the balance between these prodeath and pro-growth molecules is sphingosine kinase 1 (SK1). Our preliminary results show increased ceramide levels and decreased sphingosine kinase 1 levels in response to the induction of p53 via DNA damage. Thus, regulation of bioactive sphingolipid levels may be a key component in the p53 DNA damage response and the interaction of these pathways warrants further investigation. By understanding the details of these pathways we will be able to identify targets of therapeutic interest. The goal of this project is to determine the role of SKI in mediating the p53-dependent DNA damage response. This project will utilize wild type and p53 knockout mouse embryonic fibroblasts to investigate alterations in sphingolipid levels that are dependent on the induction of p53 by genotoxic stress. A mouse model will also be used to evaluate how these signaling mechanisms pertain to cancer development in vivo. The aims of this study are 1) determine the effects of genotoxic stress and p53 induction on the SK1/S1P pathway; 2) determine the mechanism and manifestations of p53 dependent SKI degradation in the cell; and 3) evaluate the connection between the p53 pathway and SKI/SIP pathway in vivo. This project represents an incremental step in achieving our long-term goal of determining the mechanism of involvement of sphingolipids in the cellular stress response and in organismal cancer development. PUBLIC HEALTH RELEVANCE: When human cells experience excessive stress that results in damage to their DNA, p53 levels go up to prevent the cell from passing on damaged genetic Information which could lead to cancer. This project seeks to determine how p53 regulates bioactive signalling lipids to achieve this function. This work could eventually lead to the development of more efficient cancer therapeutics with less severe side effects.